Insulin-degrading enzyme (IDE) is a zinc-metalloprotease that is involved in the clearance of insulin and amyloid-p, two key proteins for the development of diabetes and Alzheimer's disease, respectively. Additionally, IDE can bind certain opioid peptides, which function mainly in pain neurophysiology. Protease inhibitors could be used to treat pain because they may increase the lifetime of opioid peptides in vivo and there is a decreased risk for the development of addiction. Furthermore, upon opioid peptide-binding, IDE is selectively activated towards amyloid-p degradation but inhibited towards insulin hydrolysis. However, no crystal structure exists of IDE in complex with any member of the opioid peptide family;thus, I propose to solve the crystal structures of IDE in complex with a representative of each class of opioid peptide, enkephalins, dynorphins, and endorphins. I also plan to characterize the kinetic parameters and the cleavage sites of each opioid peptide by IDE. Moreover, I propose to examine whether IDE can affect the metabolism of opioid peptides and whether it is involved in opioid-mediated signaling in model cultured cells. The characterization of the interaction of opioid peptides with IDE may reveal clues about the conformational changes IDE undergoes upon opioid peptide binding that promote the selective activation toward Ap clearance and may provide a basis for the development of a new class of inhibitors of IDE designed to exclusively catabolize certain peptide substrates. Accumulating evidence strongly suggests that oxidation and nitrosylation are factors involved in the development of diabetes, Alzheimer's disease and cardiovascular disease partially due to post-translational modification of enzymes. In this application, I also propose to assess the vulnerability of IDE to oxidative and nitrosative modification and its effects on enzymatic activity. I will examine whether oxidative/nitrosative inhibition of IDE can occur in cultured neuroblastoma cells, N2a, which overexpress (3-amyloid precursor protein. Site-directed mutagenesis will be used to identify the residues that are modified. I also propose to perform X-ray crystallography to identify the structural implications of IDE inactivation by oxidation and nitrosylation. Success in this aim will define the molecular basis for oxidation and nitrosylation of IDE and may serve as a tool for the design of a therapeutic strategy to reserve IDE activity for the effective clearance of peptides like Ap and insulin. PUBLIC HEALTH RELEVANCE: Insulin-degrading enzyme is considered to be an exciting possible target for treatment of diabetes, Alzheimer's disease and cardiovascular disease. The proposed research may provide an approach to treating these ailments by manipulating the activity of this critical enzyme.